Name: implantation and recording of wireless electroretinogram and visual evoked potential in conscious rats
Uploaded: 2016-06-29T15:00:00.000+00:00
Duration: 11 min 22 s
Description: Watch this Scientific Journal Video about Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats at JoVE.com

JC would like to acknowledge the David Hay Memorial Fund, The University of Melbourne for financial support in writing this manuscript. Funding for this project was provided by an ARC Linkage grant 100200129 (BVB, AJV, CTON).

Ethics statement: Animal experiments were conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (2013). Animal ethics approval was obtained from the Animal Ethics Committee, University of Melbourne.&#160;The materials herein are for laboratory experiments only, and not intended for medical or veterinary use.
1. Preparing Electrodes
Note: A three channel transmitter is used for surgical implantation which enables 2 ERG and 1 VEP recording to be conducted simultaneously. The three active and three inactive electrodes need to be pre-fashioned into a ring shape before implantation in order to attach to the eye. For identification purposes, the manufacturer has enclosed active electrodes in half white, half colored plastic sheaths while inactive electrodes are covered in full colored sheaths. The ground electrode (clear plastic sheath) is left unaltered. For all active and inactive electrodes conduct steps 1.1, 1.2, 1.3 and 1.7.
<ol>
	<li>Untwist the double stranded stainless steel electrode with two fine tipped pliers.</li>
	<li>Trim one of the stainless steel strands (approximately 1 cm from the tip), leaving a single longer straight strand remaining to shape the ring electrode.</li>
	<li>Fold the single stainless steel strand back onto itself and twist, forming a smooth ring at the tip of the electrode.</li>
	<li>For the ERG active electrodes fashion this loop ~ 0.2 - 0.5 mm in diameter by twisting the base of the loop (for the purpose described here, shape two active electrodes in this way to record ERG from both eyes), and for the ERG inactive and VEP electrodes make the loop diameter ~ 0.8 mm diameter (in this example, do this for one active VEP electrode and all three inactive electrodes).</li>
	<li>Hook the circular VEP active electrode around a stainless steel screw (diameter 0.7 mm, length 3 mm) so the electrode rests against the screw head.</li>
	<li>Hook the 3 inactive electrodes (2 ERG, 1 VEP) around a second stainless steel screw (diameter 0.7 mm, length 3 mm).</li>
	<li>Pull the plastic sleeve forward over the sharp ends of the two stainless steel strand to reduce irritation.</li>
	<li>Sterilize the telemetry transmitters by soaking in 2% glutaraldehyde for more than 10 hr at approximately 25 &#176;C. Then rinse the transmitter with sterile saline 3 times.</li>
</ol>
2. Transmitter Implantation
<ol>
	<li>Animal Preparation
	<ol>
		<li>Disinfect&#160;the surgical area prior to experimentation by cleaning with 70% ethanol. Autoclave all surgical equipment before use and maintain equipment in chlorhexidine when not in use during surgery. Cover the animal with a surgical drape during surgery to maintain a sterile environment. Ensure all experimenters wear surgical masks, sterile&#160;gloves and gowns.</li>
		<li>Induce anesthesia with 1.5 - 2% isoflurane, at a flow rate of 3 L/min and maintained at 1.5 - 2 % at 2 L/min throughout the surgery. Confirm sufficient depth of anesthesia by absence of a pedal reflex upon pinching the muscle between the toes.</li>
		<li>Shave a 40 mm x 30 mm area over the abdomen from above the groin to the sternum.</li>
		<li>Shave a 30 mm x 20 mm area over the forehead, posterior to the eyes and anterior to the ears.</li>
		<li>Disinfect the two shaved areas. For the forehead area disinfect with 10% povidone-iodine three times (avoid the use of alcohol-based antiseptics for area near the eye, being consistent with the Standard of Practice set out by the Association of Surgical Technologists). Over the abdomen disinfect with 10% povidone iodine and 70% ethanol.</li>
		<li>Apply 1 drop of proxymetacaine to the cornea for additional topical anesthesia.</li>
		<li>Apply 1 drop of carboxymethylcellulose sodium to the cornea to prevent drying of the eyes.</li>
	</ol>
	</li>
	<li>Surgical implantation
	<ol>
		<li>Make a 10 mm incision on the head along the vertical midline between the ears with a surgical scalpel.</li>
		<li>Make a 5 mm incision on the abdomen through the skin layer along the midline below the sternum.</li>
		<li>Tunnel a 5 mm diameter cannula subcutaneously from the abdomen incision to the head incision.</li>
		<li>Feed the electrode wires (3 active and 3 inactive) of the transmitter through the cannula from the abdomen to the head.</li>
		<li>Leave the reference electrode with the transmitter base and cover the electrode tip with aseptic gauze.</li>
		<li>Cover the electrode tips (3 active and 3 inactive) with aseptic gauze.</li>
		<li>Secure the rat&#39;s head to a stereotaxic platform.</li>
		<li>Extend the forehead incision to 30mm in length with surgical scissors.</li>
		<li>Expose surgical area by retracting loose skin with 2 sutures (3 - 0) at ~ 3 and 9 o&#39;clock.</li>
		<li>Scrape off the periosteum overlying the skull using sterilized gauze to expose bregma, lambda and midline sutures.</li>
		<li>Drill two holes through the skull at the VEP active (7 mm ventral to bregma 3 mm lateral to midline) and inactive (5 mm rostral to bregma on the midline) stereotaxic co-ordinates.</li>
		<li>Attach VEP active and inactive electrodes with pre-attached stainless steel screws (diameter 0.7 mm, length 3 mm) to the skull with a small screw-driver to ~ 1 mm in depth into the premade holes. This anchors the screw to the bone without damaging the underlying cortical tissue.</li>
		<li>To implant the ERG active electrodes use an 8 - 0 suture to temporarily retract the upper eyelid.</li>
		<li>Insert a 16 to 21 G cannula subcutaneously from behind the eye through to the superior conjunctival fornix.</li>
		<li>Remove the guiding needle.</li>
		<li>Feed the active electrode through the shortened plastic catheter from the forehead towards the eye. Then remove the plastic catheter.</li>
		<li>Use a temporary suture (8 - 0), which is threaded through the electrode loop, to prevent the electrode from retracting back into the tunnel.</li>
		<li>Make a 0.5 mm incision on the superior conjunctiva at 12 o&#39;clock, 1 mm behind limbus. Use blunt dissection to expose the underlying sclera.</li>
		<li>Implant an 8 - 0 or 9 - 0 suture immediately behind the limbus at half scleral thickness.</li>
		<li>Remove temporary suture from ERG active electrode.</li>
		<li>Anchor the ERG active electrode to the half scleral thickness suture by tying 3 consecutive knots ensuring the tip of the electrode is situated close to the limbus.</li>
		<li>Close the conjunctival flap using 1 to 2 interrupted sutures (8 - 0 to 9 - 0). Ensure that the conjunctiva completely covers the ERG electrode to improve comfort.</li>
		<li>Remove the eyelid retracting suture.</li>
		<li>Repeat the procedure for the contralateral eye.</li>
		<li>Apply cyanoacrylate gel over the skull to secure all stainless screws and electrode wires. Ensure the ERG active electrodes are not pulled too tight before securing to enable eye movements.</li>
		<li>Close the head wound using a non-absorbable 3 - 0 suture.</li>
		<li>Rotate rodent to expose abdominal area. Lengthen the abdominal dermal incision to 40 mm along the linea alba with surgical scissors.</li>
		<li>Make a 35 mm incision through the inner muscle wall to expose the inner abdominal cavity.</li>
		<li>Using two sutures (3 - 0) attach the transmitter body to the animal&#39;s right hand side inner abdominal wall. Avoid contacting the liver.</li>
		<li>Loop the ground electrode and secure in this shape with a suture (3 - 0). Place it free-floating in the abdominal cavity.</li>
		<li>Close the peritoneum using a continuous suture (3 - 0).</li>
		<li>Close the skin incision using interrupted sutures (3 - 0).</li>
	</ol>
	</li>
	<li>Post-operative care
	<ol>
		<li>Monitor the animal until it has regained sufficient consciousness to maintain sternal recumbency. House the animal singly following the surgery.</li>
		<li>Administer carprofen subcutaneously for analgesia (5 mg/kg) once a day for 4 days.</li>
		<li>Add prophylactic oral antibiotics (Enrofloxin, 5 mg/kg) to the drinking water for 7 days post-surgery.</li>
		<li>Apply an anti-inflammatory ointment to skin incision sites to reduce irritation for the first 7 days post-surgery.</li>
	</ol>
	</li>
</ol>
3. Conduct ERG and VEP Recordings in Conscious Rats&#160;
<ol>
	<li>Dark adapt animal for 12 hr prior to ERG and VEP recordings</li>
	<li>Conduct all experimental manipulations under dim red illumination (17.4 cd.m-2, &#955;max = 600 nm)</li>
	<li>Apply topical anesthesia (0.5% proxymetacaine) and dilating (0.5% tropicamide) drops to the cornea.</li>
	<li>Guide the conscious rodent into a custom made, clear restrainer. 
	Note: The length of this plastic tube can be adjusted to accommodate different sized rats with the overall diameter fixed at 60 mm. The front end of the device is tapered to minimize head movement and contains perforations to enable normal breathing. This tapered front allows alignment and stabilization of the rat&#39;s head and eyes to the opening of the Ganzfeld sphere. Note that the rodent has been acclimatized to the restrainer (3 to 5 occasions) prior to surgery.</li>
	<li>Place the rodent in front of the Ganzfeld bowl with the eyes aligned with the opening of the bowl.</li>
	<li>Turn on indwelling transmitter by passing a magnet within ~ 5 cm of the transmitter. Verify that the transmitter is on by checking the LED status light on the receiver base.</li>
	<li>Collect signals over a range of luminous energies (i.e., -5.6 to 1.52 log cd.s.m-2) as described previously21. Briefly, average more signals at the dimmer light levels (~ 80 repeats) and less at the brighter luminous energies (~ 1 repeat). Gradually lengthen the interstimulus interval from 1 to 180 seconds from dimmest to the brightest light level.</li>
	<li>To isolate the ERG rod and cone responses utilize a twin-flash paradigm8. For example, initiate two flashes at 1.52 log cd.s.m-2 with a 500 msec inter-stimulus interval in-between.</li>
	<li>To record VEP signals, average 20 repeats at the brighter luminous energies (i.e., 1.52 log cd.s.m-2, 5 sec inter-stimulus interval).</li>
	<li>To evaluate implant stability, which is assessed by signal variability over time, conduct ERG and VEP recordings 7, 10, 14, 21 and 28 days post-surgery.</li>
	<li>Following experimental period, euthanize rats via intracardial injection of pentobarbiturate (1.5 ml/kg) after ketamine:xylazine anesthesia (12:1 mg/kg).</li>
</ol>

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RG and RF are employees to the commercial funder of this research (Pfizer Neusentis and Pfizer Global Research). MI was an employee of Pfizer Global Research during this research and is currently an employee of Proteostasis Therapeutics (Cambridge, USA).

Due to the minimally invasive nature of visual electrophysiology, ERG and VEP recordings in human patients are conducted under conscious conditions and only require the use of topical anesthetics for electrode placement. In contrast, visual electrophysiology in animal models is conventionally conducted under general anesthesia to enable stable electrode placement by eliminating voluntary eye and body movements. However, commonly used general anesthetics alter the ERG and VEP responses as shown by our previous publication24 and others25-27. As such development of a conscious ERG and VEP platform in a rodent model provides superior representation of physiological responses in animal models, which may in turn afford better translatability from preclinical to clinical findings. Another disadvantage of using anesthesia is that it limits the duration of an experiment. More specifically, the use of prolonged anesthesia as well as repeated administration of anesthetics can increase chance of adverse effects such as drug build up and associated respiratory problems28.
This study showed that the telemetry system in conscious rats returned robust ERG and VEP signal stability for at least 28 days post-surgery. Our group is the first to conduct conscious wireless ERG and VEP responses simultaneously24 and this manuscript details the surgical and recording procedures involved. Comparison to other surgical procedures conducted with wired conscious ERG and VEP recordings illustrate superior stability in the ERG and equivalent repeatability in VEP recordings over a 1 month period15.
The surgical techniques and subsequent conscious recordings have the potential to be applied to various animal models. The platform has potential utility in multiple applications where it is beneficial to avoid confounds associated with anesthesia29. These include drug discovery, improved translation to human studies, and chronic or longitudinal experiments.
Possible modifications to the technique include altering the number of biopotential channels implanted and simultaneously recorded. This can vary from 1 to 4 biopotential leads and thus could measure visual evoked electrophysiology from between 1 eye to 2 eyes and 2 visual cortices. Note that the alteration in the number of biopotentials channels also leads to modification of the band-width recorded which will have implications for high frequency electrophysiological signals. For example the 3 channel biopotential transmitter used in this study (F50-EEE) was chosen to show that it is possible to simultaneously record visually evoked responses from the retina and visual cortex of a conscious rat. However, these 3 channel transmitters have a band-width of 1 - 100 Hz, which can faithfully record ERG a- and b-waves but will alter oscillatory potentials due to their higher frequency24. In contrast, if it was of interest to the study to record oscillatory potentials then a transmitter with less recording channels (i.e., broader band-width) could be employed. It is also possible for the light stimulus to be altered, for example instead of conducting full-field ERG and VEP, visual physiology in response to flicker stimuli may also be utilized.
One major limitation in translating this technique to other animal models is the size of the animal&#39;s eye. One should have no problem implanting the ocular electrodes to animals bigger than rats. However, it would be challenging to implant the ERG electrode onto a mouse eye due to the smaller working area. The cortical implantation, on the other hand, should be relatively straightforward to perform in most laboratory animals.
There are several aspects of the surgery that need to be closely observed to ensure successful implantation. It is imperative that the ERG electrode ring is formed into a smooth ring due to irritation that may be induced by any rough edges on the loop. The implantation of ERG active electrodes is facilitated by two concurrent experimenters, one to stabilize the eye while the other attaches the electrode to the sclera. Particular care has to be taken to ensure the scleral suture (2.2.19) is only half-thickness, as a full thickness scleral suture will puncture the eyeball and cause vitreous leakage. The implantation of electrodes onto the skull (VEP active and ERG/VEP inactive electrodes) is less technically demanding than that of ERG electrodes. Nevertheless, it is imperative that once the electrodes are anchored to the skull, the wires are allowed to uncurl naturally to reduce any unnecessary tension. Acclimatization to the recording restrainer prior to surgical implantation is advantageous to reduce excessive movements during ERG and VEP recordings.

The ERG and VEP are minimally invasive in vivo tools to assess the integrity of retinal and visual pathways respectively in both the laboratory and clinic. The full-field ERG yields a characteristic waveform which can be broken down into different components, with each element representing different cell classes of the retinal pathway1,2. The classic full-field ERG waveform consists of an initial negative slope (a-wave), which has been shown to represent photoreceptor activity post light exposure2-4. The a-wave is followed by a substantial positive waveform (b-wave) which reflects electrical activity of middle retina, predominantly the ON-bipolar cells5-7. Furthermore, one can vary luminous energy and inter-stimulus-interval to isolate cone from rod responses8.
The flash VEP represents electrical potentials of the visual cortex and brain stem in response to retinal light stimulation9,10. This waveform can be broken down into early and late components, with the early component reflecting activity of neurons of the retino-geniculo-striate pathway11-13 and the late component representing cortical processing performed in various V1 laminae in rats11,13. Therefore simultaneous measurement of the ERG and VEP returns comprehensive assessment of the structures involved in the visual pathway.
Currently, in order to record electrophysiology in animals, anesthesia is employed to enable stable placement of electrodes. There have been attempts to measure ERG and VEP in conscious rats14-16 but these studies employed a wired setup, which can be cumbersome and may lead to animal stress by restricting animal movement and natural behavior17. With recent advances in wireless technology including improved miniaturization and battery life, it is now possible to implement a telemetry approach for ERG and VEP recording, decreasing the stress associated with wired recordings and improving long term viability. Fully internalized stable implantations of telemetry probes have proven to be successful for chronic monitoring of temperature, blood pressure18, activity19 as well as electroencephalography20. Such advances in technology will also assist with repeatability and stability of conscious recordings, increasing the platform&#39;s utility for chronic studies.

<table><tbody><tr><td>Bioamplifier</td><td>ADInstruments</td><td>ML 135</td><td>Amplifies ERG and VEP signals</td></tr><tr><td>Carboxymethylcellulose sodium 1.0%</td><td>Allergan</td><td>CAS 0009000-11-7</td><td>Maintain corneal hydration during surgery</td></tr><tr><td>Carprofen 0.5%</td><td>Pfizer Animal Health Group</td><td>CAS 53716-49-7</td><td>Post-surgery analgesia, given with injectable saline for fluid replenishment</td></tr><tr><td>Chlorhexadine 0.5%</td><td>Orion Laboratories</td><td>27411, 80085</td><td>Disinfection of surgical instrument</td></tr><tr><td>Cyanoacrylate gel activator</td><td>RS components</td><td>473-439</td><td>Quickly dries cyanoacrylate gel</td></tr><tr><td>Cyanocrylate gel&amp;nbsp;</td><td>RS components</td><td>473-423</td><td>Fix stainless screws to skull</td></tr><tr><td>Dental burr</td><td>Storz Instruments, Bausch and Lomb</td><td>E0824A</td><td>Miniature drill head of ~ 0.7 mm diameter for making a small hole in the skull over each hemisphere to implant VEP screws</td></tr><tr><td>Drill</td><td>Bosch</td><td>Dremel 300 series</td><td>Automatic drill for trepanning</td></tr><tr><td>Enrofloxin</td><td>Troy Laboratories</td><td></td><td>Prophylactic antibiotic post surgey</td></tr><tr><td>Ganzfeld integrating sphere</td><td>Photometric Solutions International</td><td></td><td>Custom designed light stimulator: 36 mm diameter, 13 cm aperture size</td></tr><tr><td>Gauze swabs</td><td>Multigate Medical Products Pty Ltd</td><td>57-100B</td><td>Dries surgical incision and exposed skull surface during surgery</td></tr><tr><td>Isoflurane 99.9%</td><td>Abbott Australasia Pty Ltd</td><td>CAS 26675-46-7</td><td>Proprietory Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery</td></tr><tr><td>Kenacomb ointment</td><td>Aspen Pharma Pty Ltd</td><td></td><td>To reduce skin irritation and itching after surgery</td></tr><tr><td>Luxeon LEDs</td><td>Phillips Lighting Co.</td><td></td><td>For light stimulation, twenty 5 W and one 1 W LEDs, controlled by Scope software</td></tr><tr><td>Needle (macrosurgery)</td><td>World Precision Instruments</td><td>501959</td><td>for suturing abdominal and head surgery, used with 3 - 0 suture, eye needle, cutting edge 5/16 circle Size 1, 15 mm</td></tr><tr><td>Needle holder (macrosurgery)</td><td>World Precision Instruments</td><td>500224</td><td>To hold needle during abdominal and head surgery</td></tr><tr><td>Needle holder (microsurgery)</td><td>World Precision Instruments</td><td>555419NT</td><td>To hold needle during ocular surgery</td></tr><tr><td>Optiva catheter</td><td>Smiths Medical International LTD</td><td>16 or 21 G</td><td>Guide corneal active electrodes from skull to conjunctiva</td></tr><tr><td>Povidone iodine 10%</td><td>Sanofi-Aventis</td><td>CAS 25655-41-8</td><td>Proprietory name: Betadine, Antiseptic to prepare the shaved skin for surgery 10%, 500 ml</td></tr><tr><td>Powerlab data acquisition system</td><td>ADInstruments</td><td>ML 785</td><td>Acquire signal from telemetry transmitter, paired to telemetry data converter</td></tr><tr><td>Proxymetacaine 0.5%</td><td>Alcon Laboratories&amp;nbsp;</td><td>CAS 5875-06-9</td><td>Topical ocular analgesia</td></tr><tr><td>Restrainer</td><td>cutom made</td><td></td><td>Front of the restrainer is tapered to minimize head movement, length can be adjusted to accommodate different rat length, overall diameter is 60 mm.&amp;nbsp;</td></tr><tr><td>Scapel blade</td><td>R.G. Medical Supplies</td><td>SNSM0206</td><td>For surgical incision</td></tr><tr><td>Scissors (macrosurgery)</td><td>World Precision Instruments</td><td>501225</td><td>for cutting tissue on the abodmen and forhead</td></tr><tr><td>Scissors (microsurgery)</td><td>World Precision Instruments</td><td>501232</td><td>To dissect the conjunctiva for electrode attachment</td></tr><tr><td>Scope Software</td><td>ADInstruments</td><td>version 3.7.6</td><td>Simultaneously triggers the stimulus via the ADI Powerlab system and collects data</td></tr><tr><td>Shaver</td><td>Oster</td><td>Golden A5</td><td>Shave fur from surgical areas</td></tr><tr><td>Stainless streel screws&amp;nbsp;</td><td>MicroFasteners</td><td>L001.003CS304</td><td>0.7 mm shaft diameter, 3 mm in length&amp;nbsp;</td></tr><tr><td>Stereotaxic frame</td><td>David Kopf</td><td>Model 900</td><td>A small animal stereotaxic instrument for locating the implantation landmarks on the skull</td></tr><tr><td>Surgical drape</td><td>Vital Medical Supplies</td><td>GM29-612EE</td><td>Ensure sterile enviornment during surgery</td></tr><tr><td>Suture (macrosurgery)</td><td>Ninbo medical needles</td><td>3-0</td><td>for suturing abdominal and head surgery, sterile silk braided, 60 cm</td></tr><tr><td>Suture needle (microsurgery)</td><td>Ninbo medical needles</td><td>8-0 or 9-0</td><td>for ocular surgery including, suturing electrode to sclera and closing conjunctival wound, nylon suture, 3/8 circle 1 &amp;times; 5, 30 cm</td></tr><tr><td>Telemetry data converter&amp;nbsp;</td><td>DataSciences International</td><td>R08</td><td>allows telemetry signal to interface with data collection software</td></tr><tr><td>Telemetry Data Exchange Matrix</td><td>DataSciences International</td><td></td><td>Gathers data from transmitters, pair with receiver</td></tr><tr><td>Telemetry data receiver</td><td>DataSciences International</td><td>RPC-1</td><td>Receives telemetry data from transmitter</td></tr><tr><td>Telemetry transmitter</td><td>DataSciences International</td><td>F50-EEE</td><td>3 channel telemetry transmitter</td></tr><tr><td>Tropicamide 0.5%</td><td>Alcon Laboratories&amp;nbsp;</td><td></td><td>Iris dilation</td></tr><tr><td>Tweezers (macrosurgery)</td><td>World Precision Instruments</td><td>500092</td><td>Manipulate tissues during abdominal and head surgery</td></tr><tr><td>Tweezers (microsurgery)</td><td>World Precision Instruments</td><td>500342</td><td>Manipulate tissues during ocular surgery</td></tr></tbody></table>

implantation and recording of wireless electroretinogram and visual evoked potential in conscious rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

The photoreceptor response is analyzed by fitting a delayed Gaussian to the leading edge of the initial descending limb of the ERG response at the top 2 luminous energies (1.20, 1.52 log c.s.m-2) for each animal, based on the model of Lamb and Pugh22, formulated by Hood and Birch23. This formula returns an amplitude and a sensitivity parameter, (Figure 1C and 1D, respectively). A hyperbolic function was fitted to the luminous energy response of rod bipolar cells for each animal, which also returned an amplitude and a sensitivity parameter, (Figure 1E and 1F respectively). Cone bipolar cell amplitude was analyzed as peak response of the waveform (top waveform of Figure 1A and 1B), with implicit time taken as the time it took to reach peak response. For further details please see Charng et al. 24.
Figure 1A and B shows ERG waveform &#177; SEMs (n = 8) in conscious rats at day 7 and 28 post-surgery. The waveforms appear to be slightly larger at day 28 compared to day 7, but linear mixed model analysis revealed no significant time effect (p = 0.14 to 0.67) for photoreceptor (dark-adapted PIII) amplitude (Figure 1C) and sensitivity (Figure 1D); rod bipolar cell (dark-adapted PII) amplitude (Figure 1E) and sensitivity (Figure 1F); cone bipolar cell (light-adapted PII) amplitude (Figure 1G) and implicit time (Figure 1H). Similarly, VEP waveform SEMs (n = 8, Figure 2A) appear comparable at 7 and 28 days post-surgery, with amplitude (Figure 2B and 2C) and timing (Figure 2D - 2F) parameters showing no significant time effect (p = 0.20 to 0.93). These results indicate robust ERG and VEP signal stability.
Average signal-to-noise (SNR, n = 8) ratio of both ERG (Figure 3A) and VEP (Figure 3B) returned good stability over the five conscious recording sessions. In this scenario, ERG signal is defined as the as the amplitude of the ERG P2 response while noise is the maximal peak to trough amplitude calculated from a 10 msec pre-stimulus interval. In the VEP, P2-N1 amplitude is deemed as the signal while noise is also returned by the peak to trough of the 10 msec pre-stimulus interval. There was no significant time effect across the SNR of both ERG and VEP (p = 0.49 and 0.62 respectively).
<img alt="Figure 1" src="/files/ftp_upload/54160/54160fig1.jpg" /> 
Figure 1: Conscious Electroretinograms Exhibit Characteristic Waveforms and Repeatable Measurements. (A-B) ERG waveforms &#177; SEMs (n = 8) across a wide range of luminous energies at day 7 (A) and 28 (B) post-surgery. (C-F) Rod and cone ERG parameters are plotted against time after implantation. Rod (dark-adapted PIII) photoreceptoral amplitude (C) and sensitivity (D), rod bipolar cell (dark-adapted PII) amplitude (E) and sensitivity (F), and cone bipolar cell (light-adapted PII) amplitude (G) and implicit time (H) all showed stable recordings over the 5 sessions. All symbols indicate average value (&#177; SEM). This figure has been modified from Charng et al.24 Figure 4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54160/54160fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54160/54160fig2.jpg" /> 
Figure 2: Conscious Visual Evoked Potentials Exhibit Characteristic Waveforms and Repeatable Measurements. (A) VEP waveforms &#177; SEMs (n = 8) are plotted at day 7 and 28 post surgery. (B-F) VEP amplitude and timing parameters are assessed over 1 month after implantation. P1-N1 (B) and P2-N1 (C) amplitude as well as P1 (D), N1 (E) and P2 (F) implicit time parameters were all stable over the 5 recording sessions. All symbols indicate average value (&#177; SEM). This figure has been modified from Charng et al.24 Figure 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54160/54160fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54160/54160fig3.jpg" /> 
Figure 3: The Telemetry System Demonstrates Stable Signal-to-Noise Ratio Over Time. The signal-to-noise ratio of the (A) ERG and (B) VEP were not significantly altered over time (n = 8). All symbols indicate average value (&#177; SEM). This figure has been modified from Charng et al.24 Figure S1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54160/54160fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats at JoVE.com

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

implantation-recording-wireless-electroretinogram-visual-evoked

Nanyang Technological University

Research

JoVE Journal

Behavior

8.7K Views.  University of Melbourne.  We tonen chirurgische implantatie en recording procedures visuele elektrofysiologische signalen van het oog (electroretinogram) en hersenen (visuele evoked potential) in ratten bij bewustzijn, dat meer analoog is aan de menselijke aandoening waarbij opnamen zonder verdoving uitgevoerd worden verwart meten. 

Video: Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Neuroscience

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Preparing a Rat Model to Assess Visual Pathway Integrity Using an Electrophysiology Setup

Source: Nguyen, C. T. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54158">Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates the preparation of a rat model to study retinal health and visual pathway integrity. Visual evoked potential and electroretinography reference and active electrodes are connected to the rat, followed by aligning the platform with the rat in front of the Ganzfeld bowl for even retinal illumination and closing the Faraday cage to avoid external interference.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

Source: Haumesser, J. K., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55940">Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats.</a> J. Vis. Exp.(2017).
This video demonstrates the detailed procedure for preparing an anesthetized rat for electrophysiological recordings of brain activity from multiple sites. The scalp is dissected to access the skull, and holes are drilled at precise locations to install and secure electrodes. Tungsten microwire electrodes are then inserted into the targeted location to record the data.

Implanting a Wireless Bidirectional Microelectrode System in a Rat Brain

Source: Melo-Thomas, L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56299">A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats.</a> J. Vis. Exp. (2017)
The video demonstrates a procedure for implanting a wireless bidirectional multichannel electrode unit into a rat brain to record and stimulate multichannel neural networks.

Recording of Visual Evoked Potentials via the Skull Electrodes in a Rat Model

Source: You, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52934">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (2015).
This video demonstrates the process of measuring visual evoked potentials or VEPs in a dark-adapted, anesthetized rat with pre-implanted skull electrodes, which record the electrical signals from the brain&#39;s visual cortex in response to light stimuli.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. VEP ...

Implanting an On-Head Electrode System in a Rat Model for Real-Time Seizure Detection

Source: Koz&#225;k, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56669">Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the method of implanting an on-head electrode system in an anesthetized rat for real-time seizure detection. The procedure includes placing stimulation and recording electrodes, securing them with dental cement, and connecting them to a head-mounted amplifier.

Concurrent Electroencephalographic and Local Field Potential Recordings in an Anesthetized Rat

Source: Kang, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56447">Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent.</a> J. Vis. Exp. (2017).
This video demonstrates the procedure for concurrent recordings of co-localized electroencephalography (EEG) and local field potentials (LFPs) in an anesthetized rat. The process involves preparing the rat and attaching stimulating electrodes to its whisker pads. An EEG spider electrode is placed on the rat&#39;s skull for surface EEG measurements. At the same time, a multi-channel microelectrode is inserted into a specific brain region through a burr hole in the skull to record neural activity from a localized brain area.

Recording of Electroretinogram and Visual Evoked Potential in a Rat

Source: Charng, J. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54160">Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats.</a> J. Vis. Exp. (2016).
This video demonstrates how to record electroretinogram (ERG) and visual evoked potential (VEP) in a conscious rat. The rat, with surgically implanted electrodes, is first dark-adapted. After applying dilating drops, the animal is placed in a restrainer and positioned in a Ganzfeld bowl. The ERG and VEP signals are then recorded.

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

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